Analyzer for analyzing particle components in urine with flow cytometry

ABSTRACT

An analyzer for analyzing urine material components includes: a sheath flow cell for forming a sample stream containing the urine material components; a light source for illuminating the sample stream; a section for detecting optical information from the illuminated material component particles; and an analyzing section for analyzing the material components; the analyzing section including a parameter extracting section for extracting parameters from the detected optical information, a section for generating a distribution diagram for the material components on the basis of the extracted parameters, a section for inputting an expectative domain of a particular material component in the distribution diagram, and a warning section for giving a warning when a cluster of data points of the particular material component deviates from the expectative domain by more than a predetermined degree.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an analyzer for analyzing urinematerial components and, more particularly, to an analyzer for analyzingblood cells, casts, mucous threads and the like in urine.

2. Description of Related Art

Hitherto known as particle analyzers are optical particle countingapparatus which are adapted to determine the number of particles on thebasis of a scattergram generated by measuring forward or lateralfluorescent light and scattered light from illuminated stainedparticles, and an electrical-resistance-type particle counter which isadapted to determine the number of particles on a size-by-size basis byinserting a needle member into an orifice (see Japanese UnexaminedPatent Publication No. 4-337459 (1992) and European Unexamined PatentPublication No. 242971A2).

Where a urine sample which includes many kinds of particles (materialcomponents) is to be analyzed by means of such a prior art apparatus,however, the qualitative and quantitative analyses of the particles aredifficult because domains of the respective kinds of particles overlapwith each other in a scattergram.

SUMMARY OF THE INVENTION

In view of the foregoing, it is an object of the present invention toprovide an analyzer for analyzing urine material components which iscapable of warning of a reduction in the accuracy of the materialcomponent analysis by determining the degree of overlapping of domainsof the respective material components in a scattergram.

In accordance with the present invention, there is provided an analyzerfor analyzing urine material components which comprises: a sheath flowcell for forming a sample stream by surrounding a sample liquidcontaining particles of the urine material components with a sheathfluid; a light source for illuminating the sample stream; aphotodetector section for detecting optical information from theilluminated material component particles; and an analyzing section foranalyzing the material components on the basis of the detected opticalinformation; the analyzing section including a parameter extractingsection for extracting a plurality of parameters from the detectedoptical information, a distribution diagram generating section forgenerating a distribution diagram for the material components on thebasis of the extracted parameters, an inputting section for inputtingdiscrimination conditions for discriminating a predetermined materialcomponent from the other material components, and a warning section forwarning that data points of the other material components are possiblypresent in a domain of the predetermined material component in thedistribution diagram if the data points of the material components inthe distribution diagram do not satisfy the discrimination conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the construction of ananalyzer in accordance with one embodiment of the present invention;

FIG. 2 is a sectional view of an orifice of a flow cell of the analyzershown in FIG. 1;

FIG. 3 is a block diagram illustrating the electrical construction of ananalyzing section of the analyzer;

FIG. 4 is a block diagram illustrating the construction of a dataprocessing section shown in FIG. 3;

FIG. 5 is a flow chart for explaining an operation to be performed bythe data processing section;

FIGS. 6 to 8 are scattergrams for explaining the operation of the dataprocessing section;

FIGS. 9 to 11 are diagrams for explaining the operation of the dataprocessing section;

FIGS. 12 to 15 are scattergrams for explaining the operation of the dataprocessing section;

FIGS. 16 and 17 are scattergrams for explaining an operation to beperformed by a warning section of the analyzer;

FIG. 18 is a histogram for explaining the operation of the warningsection;

FIG. 19 is a histogram of erythrocytes;

FIG. 20 is a scattergram for explaining domains of respective materialcomponents;

FIG. 21 is a diagram illustrating a cast;

FIG. 22 is a diagram illustrating the waveform of scatter light pulses;

FIG. 23 is a diagram illustrating the waveform of fluorescent lightpulses; and

FIGS. 24 and 25 are histograms of erythrocytes in a nonhemolytic state,erythrocytes in a hemolytic state and bacteria.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Exemplary material components (particles) to be analyzed by means of theanalyzer of the present invention include blood cells, crystals, casts,epithelial cells and bacteria contained in human urine. The particles ofthe urine material components may be pretreated with a fluorescent dyeor other labeling agents. Accordingly, the analyzer may further includea pretreatment section for pretreating the particles of the urinematerial components with such a fluorescent dye or labeling agent beforea urine sample is supplied to the sheath flow cell.

The blood cells include erythrocytes and leukocytes. The erythrocytesare often observed in urine sampled from patients of nephrouretericdiseases, hemorrhagic diseases, leukemia and the like. The leukocytesare often observed in urine sampled from patients of nephric andureteric infectious diseases, nephrophthisis and the like.

The crystals include crystals of uric acid, urates and calcium oxalatecontained in acidic urine, and crystals of ammonium magnesium phosphate,calcium carbonate and ammonium urate contained in alkaline urine, andcrystals of DHA (2,8-dihydroxyadenine) which are often observed in urinesampled from patients of APRT deficiency and are likely to cause urinarycalculus.

The casts are such that hemocytes and uriniferous epitheliocytes areenclosed in a base of Tomm-Horsfall mucoprotein agglutinated inuriniferous tubules in the presence of a small amount of plasma protein(albumin). The term "cast" comes from the fact that the casts are formedin the uriniferous tubules as a casting mold. The casts are also called"cylinders" in view of their shape. The presence of casts impliestemporary occlusion of the uriniferous tubules, and is an importantsymptom suggesting a nephric disorder. Particularly, the detection ofcasts containing hemocytes, epitheliocytes and the like has an importantclinical meaning.

The epithelial cells include squamous epitheliocytes and transitionalepitheliocytes. The squamous epitheliocytes are extremely thin cells ina circular or polygonal shape exfoliated from the internal surface ofurethra. The transitional epitheliocytes are cells having various shapessuch as pare-like shape and spindle-like shape and constituting internalsurfaces of renal pelvis, urethra, bladder and internal urethralopening.

The bacteria are herein meant by various bacteria contained in urinesampled from patients of urocystitis or pyelitis, for example.

The sheath flow cell according to the present invention preferablyincludes two cells, i.e., an upper cell and a lower cell, communicatingthrough an orifice. The sheath flow cell is adapted to form a sampleliquid containing particles into a sample stream by a hydrodynamiceffect produced by surrounding the sample liquid with a sheath flow,whereby the particles in the sample liquid are passed in line throughthe orifice.

The sheath flow cell allows the sample liquid to pass through theorifice, for example, at a rate of 0.5 to 10 m/sec.

The light source projects a light beam from the outside of the flow cellto a particle passing through the orifice, a particle just entering theorifice, or a particle just exiting out of the orifice. A laser lightsource (excluding a pulse light source) adapted for continuousillumination is preferably used as the light source in combination witha condenser lens. The width of the beam (measured in a flow direction)is preferably 5 to 30 μm.

The photodetecter section detects optical information, i.e., scatterlight and fluorescent light from particles illuminated with the lightbeam, and converts the optical information into electrical pulsesignals. Usable for the photodetecter section are a photodiode, aphototransistor, a photomultiplier tube and the like.

The analyzing section is preferably comprised of microcomputer or apersonal computer including a CPU, a ROM and a RAM.

The parameter extracting section extracts, for example, a fluorescentlight intensity Fl and a scatter light intensity Fsc from peakamplitudes of the respective pulse signals indicative of the detectedfluorescent light and scatter light, and extracts a fluorescent lightemission duration (fluorescent light pulse width) Flw and a scatterlight emission duration (scatter light pulse width) Fscw from pulsewidths of the respective signals. More specifically, a peak hold circuitis employed for the extraction of the peak amplitudes, and a countercircuit is employed for the extraction of the pulse widths.

The extracted parameter data are each converted into distribution dataF(X) in a parameter space. The distribution data is represented asfrequency data F(X1, X2, . . . , Xm) at a point defined by coordinates(X1, X2, . . . , Xm) in an m-dimensional parameter space which isdefined by m (e.g., 2) parameters X1, X2, . . . , Xm selected from nparameters Xn as required. The distribution diagram generating sectiongenerates a distribution diagram (scattergram) with the parameters X1,X2, . . . , Xm plotted as the coordinates.

Where a urine sample is to be analyzed, many kinds of urine materialcomponent particles appear in a scattergram with a wide variation in thenumber thereof and with a wide variation in the form thereof (e.g.,particles of one material component may be damaged to differentdegrees). In addition, the number and form of particles of each materialcomponent are likely to change (due to proliferation of bacteria,progress of hemolyzation, or precipitation of crystals) with the lapseof time after the sampling of the urine. The special considerations tothe urine sample make it difficult to analyze the urine materialcomponents on the basis of the scattergram in comparison with theanalysis of a blood sample.

For example, erythrocytes are hardly observed in urine sampled from ahealthy person, but observed in hematuria, and the concentration thereofis higher than several dozens to several thousands /μl. The differencein the number of erythrocytes is represented as a difference in thefrequency of occurrence in a scattergram.

Urine erythrocytes are observed in various forms, e.g., those in anonhemolytic state which sustain little damage and contain innersubstances, and those in a hemolytic state which are damaged so heavilythat almost all inner substances thereof are effused. The presence oferythrocytes in difference forms is represented as a difference in thelocation of data points or as an expanse of an erythrocyte domain in ascattergram.

With the lapse of time after the urine sampling, hemolyzation mayprogress in acidic urine, bacteria may proliferate in bacteriuria, andcrystals may grow and precipitate if the urine sample is stored at a lowtemperature. This results in a complicated distribution configuration inthe scattergram in which a plurality of peaks are present in a singledistribution area (domain). Therefore, it is difficult to determine theattribution of particles of the urine material components with highaccuracy.

For example, data points of various crystals are located within theerythrocyte domain in the scattergram, thereby reducing the accuracy ofmeasurement of the erythrocyte number.

To overcome this problem, the analyzing section according to the presentinvention analyzes the measurement accuracy by extracting distributioncharacteristics of a material component regarded as the erythrocyte.Where calcium oxalate crystal is contained in the urine, for example, adomain of the calcium oxalate crystal mostly overlaps the erythrocytedomain in the scattergram. In such a case, the analyzing sectionanalyzes the measurement accuracy by detecting the domain of the calciumoxalate crystal which extends to a higher Fsc level than the erythrocytedomain. Where DHA crystal is contained in the urine, the domain of theDHA crystal extends across the erythrocyte domain. In such a case, theanalyzing section analyzes the measurement accuracy by detecting abroader erythrocyte distribution range in a histogram generated by usingone of the two parameters employed in the scattergram.

More specifically, a forward scatter light intensity Fsc and afluorescent light intensity Fl obtained by measuring the urine materialcomponents are used as the parameters for generation of the scattergram.Then, an upper limit of the forward scatter light intensity Fsc for theerythrocyte distribution in the scattergram is set. The accuracy of themeasurement of the erythrocyte number is evaluated on the basis of thefrequency or rate of data points of material components located atlevels higher than the upper limit in the scattergram.

Alternatively, a histogram is generated by using one parameter, e.g.,the fluorescent light intensity Fl, in the scattergram, and then adistribution range in the histogram is determined. The accuracy of themeasurement of the erythrocyte number is evaluated on the basis of thedistribution range.

Thus, the present invention allows for the determination of the accuracyof analysis of a particular material component on the basis of adistribution diagram.

In accordance with another aspect of the present invention, an analyzerfor analyzing urine material components comprises: a sheath flow cellfor forming a sample stream by surrounding a sample liquid containingparticles of the urine material components with a sheath fluid; a lightsource for illuminating the sample stream; a photodetecter section fordetecting optical information from the illuminated material componentparticles; and an analyzing section for analyzing the materialcomponents on the basis of the detected optical information; theanalyzing section including a parameter extracting section forextracting a parameter from the detected optical information, adistribution section for generating a histogram on the basis of theextracted parameter, and a warning section for giving a warning when theratio of a distribution range to a maximum frequency in the generatedhistogram exceeds a predetermined value.

Also, the analyzing section of the present invention may furthercomprise a function of discriminating hemolytic-state erythrocytes fromother urine material components and computing the total number oferythrocytes including the hemolytic-state erythrocytes, on the basis ofthe generated distribution diagram.

In addition, the analyzing section of the present invention may furthercomprise a function of judging whether or not a material componentparticle being analyzed is a cast, on the basis of location of a datapoint of the material component particle in the generated distributiondiagram.

The details of the above two functions are described in our copendingU.S. patent application Nos. 08/767,782 and 08/767,783, filed Dec. 17,1996, and EP patent applications entitled "ANALYZER FOR ANALYZING URINEMATERIAL COMPONENTS" corresponding to Japanese patent applications No.HEI 7(1995)-350712 and No. HEI 7(1995)-350714 filed on the same date asthe Japanese patent application corresponding to the instantapplication, and are relied upon and incorporated by reference in thisapplication.

The present invention will hereinafter be described by way of apreferred embodiment thereof with reference to the attached drawings. Itshould be noted that the present invention is not limited by theembodiment.

FIG. 1 is a schematic diagram illustrating the construction of ananalyzer according to one embodiment of the present invention. In FIG.1, there is shown a flow cell 5 including a first cell 7a, a second cell7b and an orifice 11 connecting the first and second cells 7a and 7b andhaving a cross section as shown in FIG. 2. The first cell 7a has astainless negative electrode 12, a sample nozzle 6, and a supply port 10from which a sheath fluid fed from a sheath fluid container 9 through avalve 8 is supplied into the first cell 7a. The second cell 7b has aplatinum positive electrode 13, and a drain 14. In FIG. 1, there arealso shown a suction nozzle 3 for sucking a sample liquid subjected to apretreatment such as dilution or staining in a pretreatment section 3a,a syringe 4, and valves 1 and 2. A reference numeral 26 denotes a streamof the sample liquid injected from the sample nozzle 6.

A constant DC power supply 15 is connected between the electrodes 12 and13. Provided in association with the power supply 15 is an amplifier 16for amplifying an output voltage of the power supply 15 to output theamplified voltage as a signal 29.

An optical system includes an argon laser source 17, a condenser lens18, a beam stopper 19, a collector lens 20, a light blocking plate 30having a pin hole 21, a dichroic mirror 22, a filter 23, aphotomultiplier tube 24 and a photodiode 25.

A diluent solution and a staining solution to be used for pretreatmentare prepared in the pretreatment section 3a according to theprescription described below.

    ______________________________________                                        Diluent solution                                                              ______________________________________                                        Buffer agent  HEPES         50 mM                                                           NaOH          in an amount to                                                               adjust pH at 7.0                                  Osmotic pressure                                                                            sodium propionate                                                                           in an amount to                                   compensating agent          adjust osmotic                                                                pressure at                                                                   150 mOsm/kg                                       Chelating agent                                                                             EDTA-3K       0.4 W/W %                                         ______________________________________                                    

The electric conductivity of the diluent solution is 5 mS/cm.

    ______________________________________                                        Staining solution                                                             ______________________________________                                        First dye        DiOC6(3)     400 ppm                                         Second fluorescent                                                                             EB          1600 ppm                                         dye                                                                           ______________________________________                                    

Ethylene glycol is used as a solvent.

In the pretreatment section 3a, urine 400 μl is diluted in the abovediluent solution 1160 μl, and the above staining solution 40 μl is added(dilution ratio=4) for staining the urine material components at 35° C.

When the valves 1 and 2 are opened for a predetermined period, thesample liquid (containing particles of the urine material components inaccordance with this preferred embodiment) is sucked from the suctionnozzle 3 by a negative pressure and filled in a conduit between thevalves 1 and 2.

In turn, the syringe 4 pushes out the sample liquid from the conduitbetween the valves 1 and 2 at a constant rate, thereby injecting thesample liquid from the sample nozzle 6 into the first cell 7a. At thesame time, the valve 8 is opened so that the sheath fluid is suppliedinto the first cell 7a.

Thus, the sample liquid is surrounded with the sheath fluid and narroweddown by the orifice 11 for formation of a sheath flow. The orifice 11 isformed of an optical glass (including quartz glass) and has a squareopening (d=100 to 300 μm) as shown in FIG. 2.

The formation of the sheath flow allows particles in the sample liquidto be passed one by one in line through the orifice 11. The sampleliquid and the sheath fluid passed through the orifice 11 are dischargedfrom the drain 14.

The electrical resistance between the electrodes 12 and 13 is determinedby the conductance (electrical conductivity) of the sheath fluid, theopening size (cross section) and length of the orifice 11, theconductance of the sample liquid, and the diameter of the sample liquidstream.

A constant current is passed between the electrodes 12 and 13 from theconstant DC power supply 15 to generate a DC voltage, the amplitude ofwhich is determined by the electrical resistance between the electrodes12 and 13 and the amperage of the current. When the particles are passedthrough the orifice 11, the electrical resistance across the orifice 11is changed. More specifically, only during the passage of the particles,the electrical resistance is changed, thereby pulsating the voltagegenerated between the electrodes 12 and 13. The maximum value of thepulsated voltage (peak amplitude of a pulse) is directly proportional tothe size of a particle passing through the orifice 11. The pulse isamplified by the amplifier 16 and outputted as a resistance signal(analog pulse signal) 29.

On the other hand, a laser beam emitted from the laser source 17 isnarrowed down as having an elliptical cross section by the condenserlens 18 and projected onto the sample liquid stream 26 flowing throughthe orifice 11. The minor axis of the elliptical cross section extendingin the flow direction of the sample liquid stream is substantiallyequivalent to the diameter of a particle to be measured, e.g., about 10μm, and the major axis extending perpendicular to the flow direction issufficiently greater than the particle diameter, e.g., about 100 to 400μm.

A portion of the laser beam passing through the flow cell 5 withoutimpinging on the particle in the sample liquid is blocked by the beamstopper 19. Forward scatter light and forward fluorescent light from theparticle illuminated with the laser beam are collected by the collectorlens 20, and pass through the pin hole 21 of the light blocking plate30, reaching the dichroic mirror 22.

The fluorescent light which has a greater wavelength than the scatterlight passes through the dichroic mirror 22, and then passes through thefilter 23, whereby scatter light is filtered away therefrom. Thefluorescent light is detected by the photomultiplier tube 24, whichoutputs a fluorescent light signal (analog pulse signal) 27. The forwardscatter light is reflected by the dichroic mirror 22, and then receivedby the photodiode 25, which outputs a scatter light signal (analog pulsesignal) 28.

FIG. 3 is a block diagram illustrating the electrical construction of ananalyzing section 100 which processes the fluorescent light signal 27,the scatter light signal 28 and the resistance signal 29 thus obtained.A parameter extracting section 200 includes amplifiers 31 to 33, directcurrent regenerator circuits 34 and 35, comparators 37 and 39, peak holdcircuits 38 and 50, a clock generator 52, counters 42 and 44, A/Dconverters 43, 45 and 51, and a counter control circuit 46. In FIG. 3,there are also shown a data storage 47, a data processing section 48 anda display 49.

There will next be described a signal processing operation to beperformed by the analyzing section having such a construction.

The scatter light pulse signal 28 is amplified by the amplifier 32, andthe DC component thereof is fixed by the DC regenerator circuit 35. Apulse signal S2 outputted from the DC regenerator circuit 35 is comparedwith a threshold Th1 (see FIG. 22) by the comparator 39. A period (pulsewidth) during which the threshold Th1 is exceeded is measured as ascatter light emission duration (scatter light pulse width) Fscw by thecounter 44. The peak amplitude of the scatter light signal is held bythe peak hold circuit 50, and A/D-converted by the A/D converter 51 toprovide a scatter light intensity Fsc.

The fluorescent light pulse signal 27 is amplified by the amplifier 31,and the DC component thereof is fixed by the DC regenerator circuit 34.A pulse signal S1 outputted from the DC regenerator circuit 34 iscompared with a threshold Th2 (see FIG. 23) by the comparator 37. Aperiod during which the threshold Th2 is exceeded is measured as afluorescent light emission duration (scatter light pulse width) Flw bythe counter 42. The peak amplitude of the fluorescent light signal 27 isheld by the peak hold circuit 38, and A/D-converted by the A/D converter43 to provide a fluorescent light intensity Fl.

The resistance pulse signal 29 is amplified by the amplifier 33. Thepeak amplitude thereof (pulse peak amplitude) is held by the sample holdcircuit 40, and converted into a digital value by the A/D converter 45.

The digitized output signals of the counters 42 and 44 and the A/Dconverters 43, 45 and 51 are stored in the data storage 47 and, at thesame time, sent to the data processing section 48 for determination ofattribution of the respective urine material component particles.

More specifically, the attribution of the respective particles(erythrocyte, casts such as glass cast and inclusion cast, and the like)is determined on the basis of the distribution diagrams (histogram andscattergram). The particles in each category are counted, and the numberthereof is converted on the basis of per-microliter sample liquid. Theresult and the distribution diagrams are displayed in the display 49.

FIG. 4 is a block diagram illustrating the construction of the dataprocessing section 48. An inputting section 61 is adapted topreliminarily input various conditional data such as conditional valuesand expectative domains, and comprised of a keyboard and a mouse, forexample.

A condition storing section 61a stores the inputted conditional data. Adistribution diagram generating section 62 generates distributiondiagrams, i.e., Fl-Fsc, Fscw-Fl and Fscw-Flw scattergrams and Fl, Fsc,Flw and Fscw histograms, on the basis of the parameter informationstored in the data storage 47. An extracting section 63 extractscoordinate data and domains from the distribution diagrams generated bythe distribution diagram generating section 62.

A domain determining section 64 determines domains of the respectivematerial components in the distribution diagrams generated by thedistribution diagram generating section 62. A computing section 65performs various arithmetic operations, and counts data points of amaterial component in each domain. A warning section 66 gives a warningwhen an erroneous result on the data clustering or the counting isdetected. A judging section 67 determines the kind of the materialcomponent particles in the domain. The computation results from thecomputing section 65 and the warning from the warning section 66 aredisplayed in the display 49 like the distribution diagrams generated bythe distribution diagram generating section 62.

There will next be described principal operations to be performed by thedata processing section 48.

(1) Determination of domains in scattergram

In the analyzer, domains of the respective material components aredefined in a scattergram for determination of the attribution of thedetected material component particles. An exemplary process for thedomain determination will be described with reference to the flow chartshown in FIG. 5.

When an Fl-Fsc scattergram is generated by the distribution diagramgenerating section 62 and displayed as shown in FIG. 6 (Step S1), anexpectative domain SO where the maximum frequency point in erythrocytedistribution is possibly present is read out of the condition storingsection 61a, and located in the Fl-Fsc scattergram as shown in FIG. 7(Step S2). It is noted that an operator can change the location of theexpectative domain S0 by operating the inputting section 61.

In turn, the extracting section 63 sets a threshold for distributionfrequency in the expectative domain S0, and extracts local maximumpoints P1, P2, . . . each having a higher frequency than the thresholdand neighboring points, as shown in FIG. 8 (Step S3).

As indicated by a solid dot in FIG. 9, the local maximum point P1 ismarked as a constituent point of a domain to be determined (Step S4).

The marked point (solid point) is compared with its neighboring pointsin terms of the frequency, and then points having a lower frequency aremarked as shown in FIG. 10 (Steps S5 to S7). This process sequence isrepeated until no neighboring point has a lower frequency than themarked points (Step S6). Then, the cluster of the marked points isdefined as an area S1, as shown in FIG. 11.

Where there are a plurality of local maximum points, the processsequence described above is repeated. More specifically, an area S2 forthe local maximum point P2 is defined (Steps S9 to S13). Then, combinedareas S1 and S2 are finally defined as the erythrocyte domain (StepS14).

Domains of the other material components are defined in the same manner,and the number of material component particles falling within each ofthe domains is determined by the computing section 65 and displayed inthe display 49.

As described above, the domain determining method according to thisembodiment is such that one or more local maximum points are firstdetermined and then the domain is gradually expanded by comparing thelocal maximum points with their neighboring points in terms of thefrequency. Therefore, the determination of the domain is not influencedby a complicated configuration of the domain, a large number of localmaximum frequency points in the domain, and a small population in thedomain.

Even if the distribution of the material component particles is slightlyshifted due to a change in the sensitivity of the analyzer, thedetermination of the domain is not influenced by the shift of thedistribution.

Further, coordinate data to be processed can be reduced by consolidatingthe coordinates of distribution diagrams, whereby the process issimplified for higher speed operation. Where 4×4 coordinates areconsolidated, for example, the numbers of distribution diagrams andcoordinate data can be reduced to 1/16.

(2) Analysis of erythrocytes in hemolytic state

In accordance with the present invention, erythrocytes in a hemolyticstate can be analyzed (the analysis of the hemolytic-state erythrocytesis not performed in the prior-art analyzer). The analyzing process willhereinafter be described.

An operator operates the inputting section 61 to input expectativedomains Ao, Bo and Eo where maximum frequency points fornonhemolytic-state erythrocytes, hemolytic-state erythrocytes andcryptococcoma-like eumycetes are possibly located, respectively, in anFl-Fsc scattergram as shown in FIG. 13 in the aforesaid manner. Thedomain determining section 64 defines an area A where data points of thenonhemolytic-state erythrocytes are mainly located, an area B where thescatter light intensity Fsc is lower than the data points of theerythrocytes in the area A and data points of the hemolytic-stateerythrocytes and the cryptococcoma-like eumycetes are located, and anarea E where data points of the cryptococcoma-like eumycetes alone arelocated, as shown in FIG. 13.

An expectative domain Co where the scatter light intensity Fsc is lowerthan the data points of the erythrocytes in the area A and data pointsof the hemolytic-state erythrocytes are possibly located but no datapoint of streptobacillus is present is inputted from the inputtingsection 61, as shown in FIG. 15.

In turn, an expectative domain Do where data points of the hemolyticerythrocytes and streptobacillus are possibly located is inputted in anFscw-Fl scattergram generated by the distribution diagram generatingsection 62 as shown in FIG. 14. The domain determining section 64defines an area D where data points of the hemolytic-state erythrocytesand the streptobacillus are located in the Fscw-Fl scattergram.

The computing section 65 computes the number R of the data points of thenonhemolytic-state erythrocytes in the area A, the number r1 of the datapoints of the hemolytic-state erythrocytes simultaneously belonging tothe areas C and D, the number r2 of the data points of thehemolytic-state erythrocytes in the area B, and the number Y of the datapoints of the cryptococcoma-like eumycetes in the area E.

The number r of the hemolytic-state erythrocytes and the total numberRBC of the erythrocytes are calculated from the following equations:

    r=r1+r2                                                    (1)

    RBC=R+r                                                    (2)

Where the number Y of the cryptococcoma-like eumycetes exceedsapredetermined value e (Y>e), however, there is a possibility that thedata points of the cryptococcoma-like eumycetes as well as the datapoints of the hemolytic-state erythrocytes are present in the area B.Therefore, the data points of the hemolyticstate erythrocytes in thearea B is not taken into account, and it is considered that r2=0. On theother hand, if Y≦e, it is considered that the data points in the area Bare attributable to the hemolytic-state erythrocytes.

The number r of the hemolytic-state erythrocytes changes with the lapseof time. For example, the ratio of the nonhemolytic-state erythrocytenumber R to the hemolytic-state erythrocyte number r is 80:20 in an Fschistogram (FIG. 24), but the ratio changes into 20:80 with the lapse oftime as shown in FIG. 25.

Referring to FIG. 25, a bacteria frequency distribution J significantlyoverlaps a hemolytic-state erythrocyte frequency distribution r and,hence, the number r of the hemolytic-state erythrocytes cannotaccurately be determined. More specifically, if the hemolytic-stateerythrocyte number r exceeds a predetermined level, the calculatednumber r may be erroneous.

Therefore, the area C is an area variably set from the inputting section61 in consideration of the time-related change in the number of thehemolytic-state erythrocytes.

The computing section 65 computes the ratio h of the hemolytic-stateerythrocytes from h=r/(R+r). If the ratio h is greater than apredetermined level, the hemolytic-state erythrocyte frequencydistribution overlaps the bacteria frequency distribution. Therefore,the warning section 66 judges that the data clustering is erroneous, anddisplays a warning in the display 49.

As describe above, the analyzer of the present invention is capable ofdetermining the number of the hemolytic-state erythrocytes (which is notdetermined in the prior art), allowing for the determination of thetotal number of the urine erythrocytes. Further, the number of thehemolytic-state erythrocytes can accurately be determined inconsideration of the time-related change thereof.

(3) Warning against erroneous analysis (erroneous data clustering)

The analyzer has a function for warning against an erroneous analysis(erroneous data clustering) as described above. In addition, when it isdetermined that data points of different material components are presentin the same domain, the analyzer gives a warning indicative ofimpossibility of data clustering. An explanation will be given to thewarning process, taking as an example the data clustering for thecalcium oxalate crystal and the erythrocyte.

The distribution diagram generating section 62 generates an Fl-Fscdistribution diagram (two-dimensional scattergram) and the domaindetermining section 64 determines a domain X of the calcium oxalatecrystal as shownin FIG. 16. Then, the domain determining section 64compares the domain X with the preliminarily inputted expectative domainS0 where data points of erythrocytes are possibly located. The domain Xof the calcium oxalate crystal mostly overlaps the expectativeerythrocyte domain S0, and extends to a higher Fsc level than the domainS0. If it is determined that adifference in the highest Fsc levelbetween the domains X and S0 exceeds a predetermined level, the warningsection 66 warns that the data clustering for the erythrocyte isimpossible due to the presence of the calcium oxalate crystal.

Where DHA crystal particles are present in a urine sample, a domain Y ofthe DHA crystal extends across the expectative erythrocyte domain S0 asshown in an Fl-Fsc scattergram of FIG. 17. Therefore, it is impossibleto accurately cluster the data points of the erythrocytes.

In such a case, the warning section 66 determines the ratio b/a of apeak frequency value a to a distribution range b at a frequency level ofa/5 in an Fl histogram (FIG. 18) generated by the distribution diagramgenerating section 62, and compares the ratio b/a with a predeterminedvalue. The predetermined value is set on the basis of a histogram fortypical erythrocyte distribution (FIG. 19). If the ratio b/a exceeds thepredetermined value, the warning section 66 warns that the dataclustering for the erythrocyte is impossible due to the presence of theDHA crystal.

(4) Detection and subdivision of casts

Where the urine material components (particles) are pretreated by astaining method for staining cell membranes and nuclei, the scatterlight emission duration Fscw and the fluorescent light emission durationFlw are roughly equal to each other (Fscw≈Flw) in the case of bloodcells, epitheliocytes, bacteria and crystals. In the case of castscontaining protein bodies, however, the scatter light emission durationFscw and the fluorescent light emission duration Flw are different inthe level (Fscw>Flw) because the protein bodies are less stainable.

Where casts contain inclusion bodies, only the inclusion bodies arestained and, hence, the ratio of the scatter light emission duration tothe fluorescent light emission duration varies depending on the densityof the inclusion bodies.

By utilizing such characteristics, the analyzer subdivides the castsinto two types, i.e., an inclusion cast and a glass cast (containing noinclusion).

More specifically, the length L of a cast Z as shown in FIG. 21 isdirectly proportional to the scatter light pulse width Fscw shown inFIG. 22. Where the cast Z is preliminarily stained, the lengths L1, L2and L3 of inclusion bodies Z1, Z2 and Z3 in the cast Z are directlyproportional to pulse widths Flw1, Flw2 and Flw3 of fluorescent lightsignals, respectively, as shown in FIG. 23. It is noted that "Th1" inFIG. 22 and "Th2" in FIG. 23 are predetermined thresholds of thecomparators 39 and 37, respectively, shown in FIG. 3.

In the analyzer, the relationship among Fl, Flw1, Flw2 and Flw3 isrepresented as follows:

    Fl=Flw1+Flw2+Flw3                                          (3)

Where fluorescent light pulse widths Flw1, Flw2, . . . Flwn are obtainedfor a single cast, the pulse width Flw for the cast is calculated asfollows: ##EQU1##

In an Fscw-Flw scattergram generated by the distribution diagramgenerating section 62 on the basis of the pulse width Flw thus obtained,an erythrocyte domain T1, a leukocyte domain T2 and an epitheliocytedomain T3 are located generally along a line L1, while a cast domain T4is separated from the domains T1 to T3 by a boundary line L2.

Therefore, the judging section 67 defines the domain T4 located belowthe line L2 as the cast domain.

Alternatively, the determination of the cast domain may be based on thecoordinate data in the scattergram. That is, a cluster of coordinatedata having Flw/Fscw values smaller than the inclination of the line L2may be defined as the cast domain.

In general, the amount of inclusion in a cast determines the type of thecast, i.e., the inclusion cast or the glass cast (containing noinclusion).

Therefore, the line L3 is inputted as a reference or the cast domaindetermination from the inputting section 61 as shown in FIG. 20. Thisallows the judging section 67 to define an area T4a above the line L3 asthe inclusion cast domain and an area T4b below the line L3 as the glasscast domain.

In accordance with the present invention, a warning is given when thereliability of analysis data obtained from the analysis of urinematerial components is reduced. Therefore, highly accurate data analysiscan be ensured.

What is claimed is:
 1. An analyzer for analyzing particle components inurine, comprising:a sheath flow cell for forming a sample stream bysurrounding a sample liquid containing the particle components with asheath fluid, the particle components being treated with a fluorescentdye; a light source for illuminating the sample stream; a photodetectorsection for detecting scatter and fluorescent light as opticalinformation from the illuminated particle components; and an analyzingsection for analyzing the particle components on the basis of thedetected optical information; wherein the analyzing section includes: aparameter extracting section for extracting a plurality of parametersfrom the detected optical information; a distribution diagram generatingsection for generating a distribution diagram for the particlecomponents on the basis of the extracted parameters; a domaindetermining section for clustering the particle components in thedistribution diagram to determine a domain containing desired particlecomponents; and a warning section for generating a histogram withrespect to particle components in the determined domain and for judginga configuration of the histogram to provide a warning that particlecomponents different from the desired particle components are present inthe determined domain.
 2. An analyzer as set forth in claim 1,whereinthe warning section judges the configuration of the histogram based on aratio of a distribution range to a maximum frequency of the histogram,and the warning section provides the warning when the ratio is greaterthan a predetermined value.
 3. An analyzer as set forth in claim 1,wherein the histogram is generated using an intensity of the detectedfluorescent light.
 4. An analyzer as set forth in claim 1, wherein thedesired particle components include erythrocytes.
 5. An analyzer as setforth in claim 1, wherein the different particle components includecrystals.